Ultralow-temperature storage method for azolla spores
Through the ultra-low temperature preservation method of red pudding spores, liquid nitrogen is used to preserve red pudding spores, and the conditions are adjusted during the recovery of culture, the problem that red pudding relies on nutrient preservation in the existing technology is solved, and efficient spore preservation and recovery of germination rate is achieved, and resource communication convenience and genetic stability are improved.
Patent Information
- Application Number
- CN202510045682.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-11
AI Technical Summary
In the prior art, the reproduction of red pudding depends on nutrient preservation, resulting in large resource occupation, short storage time, frequent separation of pudding, and long-term preservation will lead to species degeneration and genetic mutation.
The ultra-low-temperature preservation method of red puff spores is adopted, including spore extraction, pretreatment, ultra-low-temperature preservation, chlorination, re-wetting and restoration culture steps. The red puff spores are preserved by liquid nitrogen and the conditions are adjusted during restoration of culture.
The long-term preservation of red droops was achieved, and the average germination rate of spores after restoration and culture reached 61.5%, up to 71%. At the same time, space and manpower were saved, and the convenience of communication and genetic stability of resources were improved.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant preservation, and in particular to a method for ultra-low temperature preservation of Azolla spores. Background Art
[0002] Azolla, scientific name Azolla affine Azolla ), belongs to the phylum Pteridophyta ( Pteridophyta ) Pteridaceae ( Phylum ) Pteridophyta ( Leptosporangiatidae ) Sophorales Salviniales ) Azolla family ( Azollaceae ) Azolla Azolla ) aquatic plants. Symbiotic nitrogen-fixing cyanobacteria in its leaf cavity reproduce quickly, doubling the fresh yield in 3-5 days. It has the physiological characteristics of nitrogen fixation, hydrogen release, potassium enrichment, carbon fixation, and emission reduction. It can be used as fertilizer, feed, food, medicine, bioenergy, and ecological utilization. The reproduction of Azolla is a staggered asexual reproduction and sexual reproduction. Because most resources cannot naturally spore, they are usually preserved and exchanged in vegetative bodies. Vegetative body preservation requires a large amount of land, has a short preservation time, and requires regular separation (usually once every 15-20 days). Continuous maintenance is time-consuming and labor-intensive, with high labor costs, and is not conducive to the exchange of Azolla germplasm resources. Vegetative body preservation requires regular separation, which can easily lead to resource mixing during operation, and long-term vegetative body preservation will lead to species degeneration and genetic variation. Summary of the invention
[0003] The technical problem to be solved by the present invention is: in view of the deficiencies of the above-mentioned prior art, a method for ultra-low temperature preservation of Azolla spores is provided.
[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a method for ultra-low temperature preservation of Azolla spores, comprising the following steps: (1) Spore extraction: Extracting spore fruits from the spore-bearing Azolla; (2) Pretreatment before storage: picking out the mature female spore fruit with male spores in the spore fruit under a microscope, drying them, and obtaining dried spores; (3) Ultra-low temperature storage: Place the dried spores in cryopreservation tubes, mark them, neatly stack them in cryopreservation boxes, and directly place them in liquid nitrogen tanks for long-term storage; (4) Thawing: Take out the cryotube from the liquid nitrogen tank and directly put it into a 37°C water bath to thaw; (5) Rehumidification: After thawing, open the lid of the cryotube and place it in a saturated copper sulfate solution for rehumidification; (6) Recovery culture: Pour the rehumidified spores into 1 / 2MS-N liquid culture medium, culture in the dark for 1 to 3 days, and then resume light culture.
[0005] Furthermore, in the above-mentioned method for ultra-low temperature preservation of Azolla spores, step (1) specifically comprises: placing the dried Azolla body with spores on the top of a sieve, and using a strong water jet to separate the spore fruit from the stem, recovering the residue on the sieve, layering the residue on a 3 M sorbitol water step gradient, and centrifuging for 10 min to obtain the spore fruit suspended in the upper layer, separating the spore fruit, and washing with water by centrifugation to obtain the spore fruit.
[0006] Furthermore, in the above-mentioned method for ultra-low temperature preservation of Azolla spores, step (1) is specifically as follows: placing the dried Azolla spore-bearing bodies on top of 1000 μm, 500 μm and 200 μm sieves arranged in sequence, and using a strong water jet to separate the spore fruit from the stem, and the residue recovered on the 200 μm sieve is then layered on a 3 M sorbitol water step gradient and centrifuged at 400 g for 8 min, the spore fruit is suspended in the upper layer, the separated spore fruit is washed three times by centrifugation with 50 ml of water, and the obtained spore fruit is placed in a centrifuge tube, distilled water is added, and stored in a refrigerator at 4°C for later use.
[0007] Furthermore, in the above-mentioned method for ultra-low temperature preservation of Azolla spores, the drying in step (2) is specifically as follows: the mature female sporocarps are placed in an aluminum foil box and dried in a constant temperature box at 26° C. for 2 days.
[0008] Furthermore, in the above-mentioned method for ultra-low temperature preservation of Azolla spores, the 1 / 2MS-N liquid culture medium in step (6) is: 1.34 grams of M571 culture medium produced by PhytoTech is added to each liter of distilled water, and the pH is adjusted to 5.8.
[0009] Furthermore, in the above-mentioned method for ultra-low temperature preservation of Azolla spores, the conditions for light culture in step (6) are: light intensity 5000-6000LUX, photoperiod 12h.
[0010] The beneficial effect of the present invention is that in the method for ultra-low temperature preservation of Azolla spores of the present invention, liquid nitrogen is used to preserve Azolla spores, and the conditions for spore recovery culture after ultra-low temperature preservation are set, so that the average germination rate of Azolla spores preserved for a long time by the method of the present invention reaches 61.5% after 23 days of recovery culture, and the highest can reach 71%. It is equivalent to the germination rate of spores not treated with ultra-low temperature (61.3%). The method of the present invention saves a lot of space and manpower, and the ultra-low temperature preservation is highly safe, genetically stable, and has a long storage time, and is also convenient for the exchange of Azolla resources. DETAILED DESCRIPTION
[0011] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in conjunction with the implementation methods.
[0012] Embodiment 1 A method for ultra-low temperature preservation of Azolla spores comprises the following steps: (1) Spore extraction: The dried spore-bearing Azolla bodies were placed on top of 1000 μm, 500 μm and 200 μm sieves, and the spore fruit was separated from the stem using a strong water jet. The residue was recovered on the sieve and then layered on a 3 M sorbitol-water step gradient and centrifuged at 400 g for 10 min. The spore fruit was suspended in the upper layer. The separated spore fruit was washed three times by centrifugation with 50 ml of water.
[0013] (2) Pretreatment before storage: Before storage, pick the mature female spore fruit with male spores under a microscope, place it in an aluminum foil box, and dry it in a constant temperature box at 26°C for 2 days; (3) Ultra-low temperature storage: Place the dried spores in 5-10 ml cryotubes, mark them, neatly stack them in a cryobox, and directly place them in a liquid nitrogen tank for long-term storage; (4) Thawing: Take out the cryopreserved tube containing the long-term ultra-low temperature preserved Azolla spores from the liquid nitrogen tank and directly put it into a 37°C water bath to thaw for 5 min; (5) Rehumidification: After thawing, open the lid of the cryovial and place it in a saturated copper sulfate solution for rehumidification at room temperature for 6 hours.
[0014] (6) Recovery culture: Pour the rehumidified Azolla spores into 1 / 2MS-N liquid culture medium, culture in the dark for 1 to 3 days, and then resume light culture (light intensity 5000-6000LUX, photoperiod 12h).
[0015] The 1 / 2MS-N liquid culture medium is: 1.34 grams of ammonium nitrate-free culture medium (M571) produced by PhytoTech is added to each liter of distilled water, and the pH is adjusted to 5.8.
[0016] After long-term preservation through the above steps, the average germination rate of the Azolla spores reached 61.5% after 23 days of recovery culture.
[0017] Embodiment 2 A method for ultra-low temperature preservation of Azolla spores comprises the following steps: (1) Spore extraction: The dried spore-bearing Azolla bodies were placed on top of 1000 μm, 500 μm and 200 μm sieves arranged in sequence, and the spore fruit was separated from the stem using a strong water jet. The residue recovered on the 200 μm sieve was then layered on a 3 M sorbitol: water step gradient and centrifuged at 400 g for 8 min, with the spore fruit suspended in the upper layer. The separated spore fruit was washed three times by centrifugation with 50 ml of water. The obtained spore fruit was placed in a centrifuge tube and stored in a refrigerator at 4°C with distilled water for later use.
[0018] (2) Pretreatment before storage: Before storage, pick the mature female spore fruit with male spores under a microscope, place it in an aluminum foil box, and dry it in a constant temperature box at 26°C for 2 days; (3) Ultra-low temperature storage: Place the dried spores in 5-10 ml cryotubes, mark them, neatly stack them in a cryobox, and directly place them in a liquid nitrogen tank for long-term storage; (4) Thawing: Take out the cryopreserved tube containing long-term ultra-low temperature preserved Azolla spores from the liquid nitrogen tank and directly put it into a 37°C water bath to thaw for 4 minutes; (5) Rehumidification: After thawing, open the lid of the cryovial and place it in a saturated copper sulfate solution to rehumidify at room temperature for 5 hours.
[0019] (6) Recovery culture: Pour the rehumidified Azolla spores into 1 / 2MS-N liquid culture medium, culture in the dark for 1 to 3 days, and then resume light culture (light intensity 5000-6000LUX, photoperiod 12h).
[0020] The 1 / 2MS-N liquid culture medium in step (6) is as follows: 1.34 g of ammonium nitrate-free culture medium (M571) produced by PhytoTech is added to each liter of distilled water, and the pH is adjusted to 5.8.
[0021] After the above steps, the long-term preservation of the Azolla spores had an average germination rate of 60.5% after 23 days of recovery culture. Embodiment 3 A method for ultra-low temperature preservation of Azolla spores comprises the following steps: (1) Spore extraction: The dried spore-bearing Azolla bodies were placed on top of 1000 μm, 500 μm and 200 μm sieves, and the spore fruit was separated from the stem using a strong water jet. The residue recovered on the 200 μm sieve was then layered on a 3 M sorbitol: water step gradient and centrifuged at 400 g for 12 min, with the spore fruit suspended in the upper layer. The separated spore fruit was washed three times by centrifugation with 50 ml of water. The obtained spore fruit was placed in a centrifuge tube and added with distilled water and stored in a refrigerator at 4°C for later use.
[0022] (2) Pretreatment before storage: Before storage, pick the mature female spore fruit with male spores under a microscope, place it in an aluminum foil box, and dry it in a constant temperature box at 26°C for 2 days; (3) Ultra-low temperature storage: Place the dried spores in 5-10 ml cryotubes, mark them, neatly stack them in a cryobox, and directly place them in a liquid nitrogen tank for long-term storage; (4) Thawing: Take out the cryopreserved tube containing long-term ultra-low temperature preserved Azolla spores from the liquid nitrogen tank and directly put it into a 37°C water bath to thaw for 6 minutes; (5) Rehumidification: After thawing, open the lid of the cryovial and place it in a saturated copper sulfate solution for rehumidification at room temperature for 7 hours.
[0023] (6) Recovery culture: Pour the rehumidified Azolla spores into 1 / 2MS-N liquid culture medium, culture in the dark for 1 to 3 days, and then resume light culture (light intensity 5000-6000LUX, photoperiod 12h).
[0024] The 1 / 2MS-N liquid culture medium in step (6) is as follows: 1.34 g of ammonium nitrate-free culture medium (M571) produced by PhytoTech is added to each liter of distilled water, and the pH is adjusted to 5.8.
[0025] After long-term preservation through the above steps, the average germination rate of the Azolla spores reached 71% after 23 days of recovery culture.
[0026] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for cryopreservation of Azolla spores, characterized in that: The following steps are involved: (1) Spore extraction: Extracting spore fruits from the spore-bearing Azolla; (2) Pretreatment before storage: picking out the mature female spore fruit with male spores in the spore fruit under a microscope, drying them, and obtaining dried spores; (3) Ultra-low temperature storage: Place the dried spores in cryopreservation tubes, mark them, neatly stack them in cryopreservation boxes, and directly place them in liquid nitrogen tanks for long-term storage; (4) Thawing: Take out the cryotube from the liquid nitrogen tank and directly put it into a 37°C water bath to thaw; (5) Rehumidification: After thawing, open the lid of the cryotube and place it in a saturated copper sulfate solution for rehumidification; (6) Recovery culture: Pour the rehumidified spores into 1 / 2MS-N liquid culture medium, culture in the dark for 1 to 3 days, and then resume light culture.
2. The method for cryopreservation of Azolla spores according to claim 1, characterized in that: Step (1) specifically comprises: placing the dried spore-bearing Azolla bodies on the top of a sieve, and using a strong water jet to separate the spore fruit from the stem, recovering the residue on the sieve, layering the residue on a 3 M sorbitol-water step gradient, and centrifuging to obtain the spore fruit suspended in the upper layer, separating the spore fruit, and washing with water by centrifugation to obtain the spore fruit.
3. The method for cryopreservation of Azolla spores according to claim 1, characterized in that: Step (1) is specifically as follows: placing the dried spore-bearing Azolla bodies on top of 1000 μm, 500 μm and 200 μm sieves arranged in sequence, and using a strong water jet to separate the spore fruit from the stem, and then layering the residue recovered on the 200 μm sieve on a 3 M sorbitol-water step gradient and centrifuging at 400 g for 8 min, with the spore fruit suspended in the upper layer, and the separated spore fruit is washed three times by centrifugation with 50 ml of water, and the obtained spore fruit is placed in a centrifuge tube, added with distilled water and stored in a refrigerator at 4°C for later use.
4. The method for cryopreservation of Azolla spores according to claim 1, characterized in that: The drying in step (2) is specifically as follows: the mature female sporocarps are placed in an aluminum foil box and dried in a constant temperature box at 26° C. for 2 days.
5. The method for cryopreservation of Azolla spores according to claim 1, characterized in that: The 1 / 2MS-N liquid culture medium in step (6) is as follows: 1.34 g of M571 culture medium produced by PhytoTech is added to each liter of distilled water, and the pH is adjusted to 5.
8.
6. The method for cryopreservation of Azolla spores according to claim 1, characterized in that: The conditions for light culture in step (6) are: light intensity 5000-6000LUX, photoperiod 12h.